The metabolic fate of nucleoside analogues which possess antiviral activity plays an important role in the efficacy and toxicity of these compounds. The purpose of these studies was to investigate the metabolism of nucleoside antivirals in tissues of the eye. Topical instillation of thymidine and its analogues produced maximal drug concentrations in the conjunctival and aqueous one hour after application and two hours after application in the cornea. In rabbits which had been infected with herpes simplex virus, type I, 72 hr prior to topical treatment, the peak concentrations of analogues were about two-fold greater in the cornea, aqueous and iris than in noninfected animals. In situ, none of the analogues produced macroscopic signs of ocular toxicity in rabbits, however, in the tissue culture system the following order of toxicity was established: 9-(2-hydroxyethoxymethyl) guanine less than or equal to 5'amino-2',5' dideoxy-5-iodouridine much less than 9-beta-D-arabinofuranosyladenine less than or equal to 1-beta-D-arabinofuranosylthymine less than or equal to 9-beta-D-arabinofuranosyladenine-5'-monophosphate less than 5-iodo-2'-deoxyuridine less than 5-trifluoromethyl-2'-deoxyuridine. The anabolically formed metabolities, fluoride ions, 5-carboxy-2'-doeoxyuridine, 5-iodouracil, hypoxanthine arabinoside, and 5-carboxyuracil were not toxic to Chang conjunctival cells at the highest concentration studied. Neither the parent compounds nor their metabolites were toxic to the corneal endothelium as determined via corneal swelling and scan electron microscopy. F-ion, however, was observed to inhibit the metabolism of exogenous supplied glucose without altering the endothelial barrier.